Bacterial infections are a major cause of mortality and morbidity in the world, and new strategies for improving their treatment are greatly needed. A key issue limiting treatment of bacterial infections is an inability to rapidly determine antibioti susceptibilities. Conventional microbiological diagnosis depends on culturing from bodily specimens, often requiring 1-3 day incubation times that can be further delayed by the presence of slow- growing or finicky pathogens. The objective of this proposal is to develop a flow cytometric strategy for rapidly determining MICs of antibiotics against the six most common blood stream infection (BSI)-causing bacteria. Requiring <1-hr antibiotic incubation times to see characteristic responses, this new susceptibility strategy is designed to determine antimicrobial MICs within 4 hours of blood culture positivity, portending significant improvements to patient treatment outcomes and lowered incidence of drug resistance caused by excessive antibiotic use. The flow cytometric methods proposed here rely on statistical, scattered light and selective bacterial (vs. mammalian cell) delivery and enrichment technologies recently developed in our laboratories. The central hypothesis of this proposal is that Antimicrobial susceptibility can be generated within 4 hours of positive blood culture, for the six most common BSI-causing bacteria, using a combination of flow cytometry technologies that measure antibiotic-induced ROS and scatter changes in bacteria, each coupled with rigorous multidimensional statistical distance metrics for quantification. The experiments in this proposal will develop a complementary set of flow cytometric technologies that rapidly determine antibacterial MICs of bacteria either after subculture isolation or after being recovered directly from positive blood cultures. When successful, these studies will reduce the time to antibiotic susceptibility determinations to as little as ~4 hours, post positive blood culture. Removing this treatment bottleneck will enable actionable information to be garnered at least 36 hours faster than currently implemented, directly improving patient outcomes and minimizing antibiotic resistance se- lection. Each Aim utilizes highly innovative technologies to achieve these potential significant benefits to human health. In Aim 1,our unique maltodextrin-based bacterial targeting,30 and our highly sensitive fluorogenic ROS dosimeters31 will be combined and applied to quantify the demonstrated ROS production42-44 upon near- MIC antibiotic exposure. Aim 2 will demonstrate that <1-hr, near-MIC antibiotic exposure induces characteristic bacterial morphology changes that are detectable with scattered light. Multidimensional statistical distance metrics vs. paired controls are developed to quantify these changes and determine label-free MICs in the presence of biovariability. Aim 3 will combine the innovations of Aims 1 & 2 to probe clinical isolates using ROS and scatter with 3-D statistical distance metrics, while gating on fluorophore-targeted bacteria rapidly recovered from blood culture will further decrease time-to-result constraints. The proposed experiments address the great need to reduce the tremendous human and economic costs of bacterial infections.